In a recent feature article, Gillette and Carr1 describe targeted mass spectrometry (MS) in terms of its clinical relevance for biomedicine. In their view, the major benefit of targeted MS is its potential role in clinical proteomics and the opportunity to take proteome quantification from the research laboratory into the clinic. In this way, the researchers demonstrate the expected utility of targeted MS for both clinical diagnostics and prediction applications.
Early on, targeted MS through multiple ion monitoring relied on the time consuming extraction of a few select ions from a full MS scan to identify and quantify analytes. The development of triple quadrupole instrumentation allowed researchers to mass select and fragment precursor ions and monitor for signals to identify over 100 transitions per second in Multiple Reaction Monitoring mode. In general, MRM uses peptide and protein concentrations drawn from the peak area ratios to quantify proteins with high precision but relatively low accuracy and can be useful when monitoring relative changes among samples, such as when monitoring protein changes at various stages of disease. When absolute quantification is required, researchers use stable isotope-labeled proteins and peptides. Using a labeled, internal standard increases both experimental confidence and reproducibility across labs.
The complex nature of biological samples heightens the probability of experimental interference since the transition signal may become distorted by background noise. For biological applications, immunoaffinity captures a target protein with the assistance of antipeptide antibodies, thereby addressing this issue. Taken together, stable isotope standards and immunoaffinity enrichment comprise the SISCAPA method, which can increase the clinical versatility of the targeted MS approach, particularly when highly multiplexed. Gillette and Carr1 describe these methods as highly reproducible and note the detection limit of 1 ng of protein per milliliter of plasma and 15% or less coefficient of variation.
In terms of biomedicine, novel biomarkers stand out as a primary clinical goal of targeted MS. The heterogeneous nature of disease means that the verification step in biomarker identification or discovery is particularly important. Targeted MRM-MS may offer a time-sensitive approach to verifying candidate biomarkers. Easy multiplexing means that a single run of LC-MRM-MS can accurately quantify hundreds of analytes or up to 50 analytes by peptide immuno-MRM. Using ultra-high-performance LC to increase the flow rate results in increased robustness, while decreased selectivity can be mediated by increasing the sample and column size. Gillette and Carr1 point to Whiteaker et al.’s2 mouse model of breast cancer to demonstrate the reliability of MRM-MS for verification and cite the median coefficient of variation as 5.7% at limit of quantification (LOQ). Other examples of using MRM-MS for verification include cardiovascular disease3 and ovarian cancer.4 The immunoenrichment strategy mass spectrometric immunoassay (MSIA), when coupled with selected reaction monitoring (SRM), has been recently demonstrated to address the issue of protein heterogeneity in a way that preserves other important experimental features including high sensitivity, robustness, high throughput, and precision in clinically relevant applications that include Alzheimer’s Disease, cancer, endocrine function, neurological, and cardiovascular conditions.5
Gillette and Carr1 contend that the distinct advantages of targeted MS leave the approach poised for adoption as the go-to clinical tool for biomedicine. They foresee the development of target MS-based diagnostics and clinical predictors. The writers predict that the eventual deployment of this technology to standard hospital laboratories will require the creation of turnkey applications and intelligent software that couples well with existing instrumentation.
1. Gillette, M.A., and Carr, S.A. (2013) ‘Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry‘, Nature Methods, 10 (1), (pp. 28–34)
2. Whiteaker, J.R., et al. (2011) ‘A targeted proteomics-based pipeline for verification of biomarkers in plasma‘, Nature Biotechnolology, 29 (7), (pp. 625–634)
3. Addona, T.A., et al. (2009) ‘Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma‘, Nature Biotechnology, 27 (7), (pp. 633–641)
4. Hüttenhain, R., et al. (2012) ‘Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics‘, Science Translational Medicine, 4 (142), 142ra94
5. Krastins, B., et. al. (2013) ‘Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum‘, Clinical Biochemistry, 46(6), (pp. 399-410)